The invention relates generally to the field of prevention and treatment of infectious diseases, particularly infection by intracellular pathogens such as Mycobacterium.
The broad classification of intracellular pathogens includes viruses, bacteria, protozoa, fungi, and intracellular parasites. These virulent pathogens multiply within the cells of the infected host organism rather than extracellularly and are major causes of morbidity and fatality world-wide. For example, intracellular pathogens are responsible for an estimated 10,000,000 new cases of tuberculosis per year in the world (approximately 25,000 per year in the United States), approximately 3,000,000 deaths per year from tuberculosis, an estimated 12,000,000 cases of leprosy, and an estimated 10,000,000 cases of American trypanosomiasis (Chagas disease). Furthermore, intracellular pathogens also cause other important diseases including cutaneous and visceral leishmaniasis, listeriosis, toxoplasmosis, histoplasmosis, trachoma, psittacosis, Q-fever, and Legionellosis including Legionnaires"" disease. Few vaccines are available against such diseases and the pathogens are developing resistance to commonly used drugs.
One particular genus of intracellular bacteria, Mycobacteria, is a significant cause of morbidity and mortality, particularly among immunocompromised or elderly individuals and in countries with limited medical resources. Ninety-five percent of human infections are caused by seven species: Mycobacterium tuberculosis, M. avium (also known as the mycobacterium avium complex or M. avium-intracellulare), M. leprae, M. kansasii, M. fortuitum, M. chelonae, and M. absecessus. The most common mycobacterial infections in the United States are pulmonary infections by M. tuberculosis or M. avium. Such mycobacterial infections have been of increasing concern over the past decade, particularly in light of the increasing incidence of multi-drug resistant strains.
M. tuberculosis is the causative agent of tuberculosis, the classic human mycobacterial disease. Disease is spread by close person-to-person contact through inhalation of infectious aerosols; infection can be established if as few as one to three bacilli reach the alveolar spaces. Estimates indicates that one-third of the world""s population, including 10 million in the U.S., are infected with M. tuberculosis, with 8 million new cases and 3 million deaths reported world wide each year. Although incidence of tuberculosis steadily decreased since the early 1900s, this trend changed in 1984 with increased immigration from endemic countries and increased infection in the homeless, drug and alcohol abusers, prisoners, and HIV-infected individuals ((1995) Morbid. Mortal. Weekly Rep 44:1-87). Due to the difficulties in eradicating disease in most of these populations, tuberculosis has again threatened to pose a significant public health risk.
Mycobacterium avium is generally less of a health risk for individuals with normal immune responses; M. avium can transiently colonize these individuals, but disease due to M. avium is rare. However, M. avium infection can cause serious disease in patients having compromised pulmonary function (e.g., patients with chronic bronchitis, obstructive pulmonary disease, or pre-existing pulmonary damage (e.g., due to previous pulmonary infections or other disease). Infection in individuals having compromised pulmonary function is clinically very similar to infection by M. tuberculosis. 
M. avium infection poses the greatest health risk to immunocompromised individuals, and is one of the most common opportunistic infections in patients with AIDS (Horsburgh (1991) New Eng. J. Med. 324:1332-1338). In contrast with disease in other patients, M. avium infection can be very serious in immunocompromised individuals (e.g., AIDS patients, who have a low CD4+ T-cell count (Crowe, et al. (1991) J. AIDS 4:770-776)), and can result in disseminated infection in which virtually no organ is spared. The magnitude of such disseminated M. avium infections is overwhelming, with the bacterial load in some patients resulting in tissues that are literally filled with mycobacteria and with hundreds to thousands of bacilli per milliliter of blood. When disseminated disease occurs, M. avium infection results in considerable morbidity, and is a significant contributor to mortality in AIDS patients. Although highly active anti-retroviral therapy currently used to treat HIV-infected patients prevents the onset of M. avium infection to some extent (Autran, et al. (1997) Science. 277:112-116), this infection is extremely difficult to treat when encountered because of its poor responsiveness to anti-mycobacterial therapy (Chin, et al. (1994) J. Infect. Dis. 170:578-584; Masur (1993) New Eng. J. Med. 329:898-904).
As noted above, mycobacterial infection is normally acquired through inhalation of aerosolized infectious particles. Following inhalation, mycobacteria predominately infect and multiply within macrophages (Edwards, et al. (1986) Am. Rev. Respir. Dis. 134:1062-1071). The bacteria attach to and enter macrophages with the help of specific receptors expressed on the surface of these cells (Bermudez, et al. (1991) Infect. Immun. 59:1697-1702; Rao, et al. (1993) Infect. Immun. 61:663-670; Roecklein, et al. (1992) J. Lab. Clin. Med. 119:772-781). Studies have shown that macrophages secrete several cytokines such as tumor necrosis factor (TNF)-xcex2, interleukin (IL)-1xcex2, IL-6, granulocyte macrophage colony stimulating factor (GM-CSF), and granulocyte colony stimulating factor (Fattorini, et al. (1994) J. Med. Microbiol. 40:129-133; Newman, et al. (1991) J. Immunol. 147:3942-3948) in response to infection with mycobacteria. T cell products such as interferon (IFN)-xcex3 and IL-12 are known to be extremely important for anti-mycobacterial activity of macrophages (Fattorini, et al. (1994) J. Med. Microbiol. 40:129-133) as well as in vivo in humans and mice (Appelberg, et al. (1994) Infect. Immun. 62:3962-3971; Holland, et al. (1994) New Eng. J. Med. 330:1348-1355; Kobayashi, et al. (1995) Antimicrob. Agents Chemotherapy. 39:1369-1371).
Treatment of mycobacterial infections is complicated and difficult. For example, treatment of M. tuberculosis and of M. avium infections requires a combination of relatively toxic agents, usually three different drugs, for at least six months. The toxicity and intolerability of these medications usually result in low compliance and inadequate treatment, which in turn increases the chance of therapeutic failure and enhances the selection for drug-resistant organisms. Treatment of mycobacterial infections is further complicated in pregnant women, patients with pre-existing liver or renal diseases, and immunocompromised patients, e.g., AIDS patients.
Immunomodulatory sequences (hereinafter referred to as xe2x80x9cISSxe2x80x9d) were initially discovered in the mycobacterial genome as DNA sequences that selectively enhance NK cell activity (Yamamoto, et al. (1992) Microbiol. Immunol. 36:983-997). Uptake of mycobacterial DNA or ISS has been shown to activate cells of the innate immune system, such as NK cells and macrophages and stimulating a type-1 like response (Roman, et al. (1997) Nature Med. 3:849-854). Further, administration of ISS has been shown activate NK cells (Krieg, A et al. (1995) Nature. 374:546-549), stimulate B cells to proliferate and to produce IgM antibodies (Krieg, A et al. (1995) Nature. 374:546-549; Messina, et al. (1991) J. Immunol. 147:1759-1764; ), stimulate production of cytokines, such as IFNs, IL-12, IL-18 and TNF-xcex1 (Sparwasser, et al. (1998) Eur. J. Immunol. 28:2045-2054; Sparwasser, et al. (1997) Eur. J. Immunol. 27:1671-1679; Stacey, et al. (1999) Infect. Immun. 67:3719-3726; Stacey, et al. (1996) J. Immunol. 157:2116-2122; Halpern, et al (1996) Cell. Immunol. 167:72-78; Klinman, et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93:2879-2883) and up-regulate co-stimulatory receptors (Martin-Orozco, et al. (1999) Int. Immun. 11:1111-1118; Sparwasser, et al. (1998) Eur. J. Immunol. 28:2045-2054).
Previous studies have demonstrated the ability of immunomodulatory nucleic acid to enhance innate immunity and host survival against intracellular pathogens such as Listeria monocytogenes, Leishmania major, and Francisella tularensis (Krieg, et al. (1998) J. Immunol. 161:2428-2434; Walker, et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96:6970-6975; Zimmermann, et al. (1998) J. Immunol. 160:3627-3630; Klinman, et al. (1999) Infect. Immun. 67:5658-5663). Walker, et al. found that injection of BALB/c mice with CpG-ODN 1826 four hours after inoculation with live L. major promastigote organisms protected 65% of animals tested from progressive infection, suggesting that CpG-ODN can redirect the harmful immune response elicited by live L. major parasites and that CpG-ODN might be efficacious in the treatment of early leishmaniasis (Walker, et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96:6970-6975). Zimmnermann et al. report that single injections of CpG-ODN protected L. major-infected BALB/c mice when given during the first 8 days of infection but failed when given later. Zimmermann also found that 5 of 6 L. major-infected BALB/c mice were able to control the infection when given three consecutive doses of CpG-ODN at 5 day intervals starting on day 15 or 20 post infection (Zimmermann, et al. (1998) J. Immunol. 160:3627-3630).
In their studies with L. monocytogenes, Kreig, et al. found that IFN-xcex3 production is induced rapidly by ISS administration, returning to the basal level within 24 hours, while IL-12 (p40 and p70) is induced immediately after infection and lasts for at least 8 days (Krieg, et al. (1998) J. Immunol. 161:2428-2434). In the murine leishmaniasis model, the serum IL-12 level in the ISS-treated mice was found to be 10-fold higher than L. major-infected control mice (Zimmermann, et al. (1998) J. Immunol. 160:3627-3360).
Exogenous administration of type-1 cytokines, such as IL-12 and IFN-xcex3 increase protection against M. avium infection in humans and mice (Appelberg, et al. (1994) Infect. Immun. 62:3962-3971; Holland, et al. (1994) New Eng. J. Med. 330:1348-1355; Kobayashi, et al. (1995) Antimicrob. Agents Chemotherapy. 39:1369-1371). IFN-xcex3 and IL-12 are known to be important in host anti-mycobacterial immunity (Doherty, et al. (1997) J. Immunol. 158:4822-4831; Doherty,et al. (1998) J. Immunol. 160:5428-5435). However, administration of such cytokines is potentially dangerous to the patient, is expensive and does not provide an attractive means of preventing or treating existing infections by intracellular pathogens. Furthermore, administration of these cytokines can itself be associated with undesirable side-effects which are due at least in part to toxicity, especially at dosages sufficient to stimulate the subject""s immune system.
DNA vaccines may provide an alternative method for therapy. DNA vaccination with a plasmid which encodes M. avium antigens (65 kDa and antigen 85B) had a protective effect against M. avium infection in mice (Velaz-Faircloth, et al. (1999) Infect. Immun. 67:4243-4250). Similarly, plasmid DNA which encodes antigen 85B, ESAT-6 and MPT64 (Kamath, et al. (1999) Infect. Immun. 67:1702-1707), and hsp-65 (Bonato, et al. (1998) Infect. Immun. 66:169-175) yielded protective immunity against M. tuberculosis infection. Further DNA vaccines based upon administration of a polynucleotide encoding a mycobacterial antigen are described in WO 98/53075. However, while these methods appear promising, DNA vaccination requires identification of an antigen that will induce a protective immune response. Furthermore, the immune response elicited by these vaccines is predominantly a type-1 response (i.e., mediated by Th1 cells and primarily resulting in production of antibodies). As discussed above, a robust cellular immune type-1 immune response (i.e., an immune response mediated by Th1 cells and primarily resulting in activation of cytotoxic T cells, which secrete IFN-xcex3) is likely required to provide effective immunity against such intracellular pathogens. Finally, while DNA vaccines may provide some protection against infection in a preventive mode, their effectiveness against an ongoing infection is not proven.
There remains a need in the field for effective methods for the treatment and prevention of infection by intracellular pathogens.
The present invention features methods for treatment or prevention of infection by intracellular pathogen by administration of an immunomodulatory nucleic acid molecule (ISS). In one embodiment, ISS are administered in combination with another anti-pathogenic agent to provide a synergistic anti-pathogenic effect. In a preferred embodiment, the intracellular pathogen is Mycobacterium species.
A primary object of the invention is to provide an effective method for the prevention and/or treatment of intracellular pathogen infections in a host, particularly mycobacterial infections.
Another object of the invention is to enhance the anti-pathogenic activity, particularly the anti-mycobacterial activity, of conventional chemotherapeutics to facilitate more effective clearance of the organism from an active infection in a subject.
One advantage of the invention is that, since immunomodulatory nucleic acid molecules act through induction of the immune response of the host, the use of immunomodulatory nucleic acid molecules will not substantially result in the selection of resistant organisms. Still another advantage is that immunomodulatory nucleic acids acts in synergy with conventional antibiotics, particularly in the context of treatment of mycobacterial infection.
These and other objects and advantages will be readily apparent to the ordinarily skilled artisan upon reading the disclosure provided herein.